ResearchPad - campylobacter https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Identification of potential vulnerable points and paths of contamination in the Dutch broiler meat trade network]]> https://www.researchpad.co/article/elastic_article_14703 The poultry meat supply chain is complex and therefore vulnerable to many potential contaminations that may occur. To ensure a safe product for the consumer, an efficient traceability system is required that enables a quick and efficient identification of the potential sources of contamination and proper implementation of mitigation actions. In this study, we explored the use of graph theory to construct a food supply chain network for the broiler meat supply chain in the Netherlands and tested it as a traceability system. To build the graph, we first identified the main actors in the supply chain such as broiler breeder farms, broiler farms, slaughterhouses, processors, and retailers. The capacity data of each supply chain actor, represented by its production or trade volumes, were gathered from various sources. The trade relationships between the supply chain actors were collected and the missing relationships were estimated using the gravity model. Once the network was modeled, we computed degree centrality and betweenness centrality to identify critical nodes in the network. In addition, we computed trade density to get insight into the complexity of sub-networks. We identified the critical nodes at each stage of the Dutch broiler meat supply chain and verified our results with a domain expert of the Dutch poultry industry and literature. The results showed that processors with own slaughtering facility were the most critical points in the broiler meat supply chain.

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<![CDATA[MLST-based genetic relatedness of Campylobacter jejuni isolated from chickens and humans in Poland]]> https://www.researchpad.co/article/N2eb0d267-f054-40f4-b445-0c8d9725ee43

Campylobacter jejuni infection is one of the most frequently reported foodborne bacterial diseases worldwide. The main transmission route of these microorganisms to humans is consumption of contaminated food, especially of chicken origin. The aim of this study was to analyze the genetic relatedness of C. jejuni from chicken sources (feces, carcasses, and meat) and from humans with diarrhea as well as to subtype the isolates to gain better insight into their population structure present in Poland. C. jejuni were genotyped using multilocus sequence typing (MLST) and sequence types (STs) were assigned in the MLST database. Among 602 isolates tested, a total of 121 different STs, including 70 (57.9%) unique to the isolates' origin, and 32 STs that were not present in the MLST database were identified. The most prevalent STs were ST464 and ST257, with 58 (9.6%) and 52 (8.6%) C. jejuni isolates, respectively. Isolates with some STs (464, 6411, 257, 50) were shown to be common in chickens, whereas others (e.g. ST21 and ST572) were more often identified among human C. jejuni. It was shown that of 47 human sequence types, 26 STs (106 isolates), 23 STs (102 isolates), and 29 STs (100 isolates) were also identified in chicken feces, meat, and carcasses, respectively. These results, together with the high and similar proportional similarity indexes (PSI) calculated for C. jejuni isolated from patients and chickens, may suggest that human campylobacteriosis was associated with contaminated chicken meat or meat products or other kinds of food cross-contaminated with campylobacters of chicken origin. The frequency of various sequence types identified in the present study generally reflects of the prevalence of STs in other countries which may suggest that C. jejuni with some STs have a global distribution, while other genotypes may be more restricted to certain countries.

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<![CDATA[A Novel Mouse Model of Campylobacter jejuni Gastroenteritis Reveals Key Pro-inflammatory and Tissue Protective Roles for Toll-like Receptor Signaling during Infection]]> https://www.researchpad.co/article/5989db48ab0ee8fa60bd91ec

Campylobacter jejuni is a major source of foodborne illness in the developed world, and a common cause of clinical gastroenteritis. Exactly how C. jejuni colonizes its host's intestines and causes disease is poorly understood. Although it causes severe diarrhea and gastroenteritis in humans, C. jejuni typically dwells as a commensal microbe within the intestines of most animals, including birds, where its colonization is asymptomatic. Pretreatment of C57BL/6 mice with the antibiotic vancomycin facilitated intestinal C. jejuni colonization, albeit with minimal pathology. In contrast, vancomycin pretreatment of mice deficient in SIGIRR (Sigirr−/−), a negative regulator of MyD88-dependent signaling led to heavy and widespread C. jejuni colonization, accompanied by severe gastroenteritis involving strongly elevated transcription of Th1/Th17 cytokines. C. jejuni heavily colonized the cecal and colonic crypts of Sigirr−/− mice, adhering to, as well as invading intestinal epithelial cells. This infectivity was dependent on established C. jejuni pathogenicity factors, capsular polysaccharides (kpsM) and motility/flagella (flaA). We also explored the basis for the inflammatory response elicited by C. jejuni in Sigirr−/− mice, focusing on the roles played by Toll-like receptors (TLR) 2 and 4, as these innate receptors were strongly stimulated by C. jejuni. Despite heavy colonization, Tlr4−/−/Sigirr−/− mice were largely unresponsive to infection by C. jejuni, whereas Tlr2−/−/Sigirr−/− mice developed exaggerated inflammation and pathology. This indicates that TLR4 signaling underlies the majority of the enteritis seen in this model, whereas TLR2 signaling had a protective role, acting to promote mucosal integrity. Furthermore, we found that loss of the C. jejuni capsule led to increased TLR4 activation and exaggerated inflammation and gastroenteritis. Together, these results validate the use of Sigirr−/− mice as an exciting and relevant animal model for studying the pathogenesis and innate immune responses to C. jejuni.

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<![CDATA[A Systematic Review Characterizing On-Farm Sources of Campylobacter spp. for Broiler Chickens]]> https://www.researchpad.co/article/5989da22ab0ee8fa60b7f548

Campylobacter and antimicrobial-resistant Campylobacter are frequently isolated from broiler chickens worldwide. In Canada, campylobacteriosis is the third leading cause of enteric disease and the regional emergence of ciprofloxacin-resistant Campylobacter in broiler chickens has raised a public health concern. This study aimed to identify, critically appraise, and synthesize literature on sources of Campylobacter in broilers at the farm level using systematic review methodology. Literature searches were conducted in January 2012 and included electronic searches in four bibliographic databases. Relevant studies in French or English (n = 95) conducted worldwide in any year and all study designs were included. Risk of Bias and GRADE criteria endorsed by the Cochrane collaboration was used to assess the internal validity of the study and overall confidence in the meta-analysis. The categories for on-farm sources were: broiler breeders/vertical transfer (number of studies = 32), animals (n = 57), humans (n = 26), environment (n = 54), and water (n = 63). Only three studies examined the antimicrobial resistance profiles of Campylobacter from these on-farm sources. Subgroups of data by source and outcome were analyzed using random effect meta-analysis. The highest risk for contaminating a new flock appears to be a contaminated barn environment due to insufficient cleaning and disinfection, insufficient downtime, and the presence of an adjacent broiler flock. Effective biosecurity enhancements from physical barriers to restricting human movement on the farm are recommended for consideration to enhance local on-farm food safety programs. Improved sampling procedures and standardized laboratory testing are needed for comparability across studies. Knowledge gaps that should be addressed include farm-level drug use and antimicrobial resistance information, further evaluation of the potential for vertical transfer, and improved genotyping methods to strengthen our understanding of Campylobacter epidemiology in broilers at the farm-level. This systematic review emphasizes the importance of improved industry-level and on-farm risk management strategies to reduce pre-harvest Campylobacter in broilers.

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<![CDATA[Molecular Epidemiology of Campylobacter Isolates from Poultry Production Units in Southern Ireland]]> https://www.researchpad.co/article/5989d9d5ab0ee8fa60b6584d

This study aimed to identify the sources and routes of transmission of Campylobacter in intensively reared poultry farms in the Republic of Ireland. Breeder flocks and their corresponding broilers housed in three growing facilities were screened for the presence of Campylobacter species from November 2006 through September 2007. All breeder flocks tested positive for Campylobacter species (with C. jejuni and C. coli being identified). Similarly, all broiler flocks also tested positive for Campylobacter by the end of the rearing period. Faecal and environmental samples were analyzed at regular intervals throughout the rearing period of each broiler flock. Campylobacter was not detected in the disinfected house, or in one-day old broiler chicks. Campylobacter jejuni was isolated from environmental samples including air, water puddles, adjacent broiler flocks and soil. A representative subset of isolates from each farm was selected for further characterization using flaA-SVR sub-typing and multi-locus sequence typing (MLST) to determine if same-species isolates from different sources were indistinguishable or not. Results obtained suggest that no evidence of vertical transmission existed and that adequate cleaning/disinfection of broiler houses contributed to the prevention of carryover and cross-contamination. Nonetheless, the environment appears to be a potential source of Campylobacter. The population structure of Campylobacter isolates from broiler farms in Southern Ireland was diverse and weakly clonal.

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<![CDATA[Molecular Evidence for the Thriving of Campylobacter jejuni ST-4526 in Japan]]> https://www.researchpad.co/article/5989daa6ab0ee8fa60ba77f5

Campylobacter jejuni is a leading cause of human gastroenteritis worldwide. This study aimed at a better understanding of the genetic diversity of this pathogen disseminated in Japan. We performed multilocus sequence typing (MLST) of Campylobacter jejuni isolated from different sources (100 human, 61 poultry, and 51 cattle isolates) in Japan between 2005 and 2006. This approach identified 62 sequence types (STs) and 19 clonal complexes (CCs), including 11 novel STs. These 62 STs were phylogenetically divided into 6 clusters, partially exhibiting host association. We identified a novel ST (ST-4526) that has never been reported in other countries; a phylogenetic analysis showed that ST-4526 and related STs showed distant lineage from the founder ST, ST-21 within CC-21. Comparative genome analysis was performed to investigate which properties could be responsible for the successful dissemination of ST-4526 in Japan. Results revealed that three representative ST-4526 isolates contained a putative island comprising the region from Cj0737 to Cj0744, which differed between the ST-4526 isolates and the reference strain NCTC11168 (ST-43/CC-21). Amino acid sequence alignment analyses showed that two of three ST-4526 isolates expressed 693aa- filamentous hemagglutination domain protein (FHA), while most of other C. jejuni strains whose genome were sequenced exhibited its truncation. Correspondingly, host cell binding of FHA-positive C. jejuni was greater than that of FHA-truncated strains, and exogenous administration of rFHA protein reduced cell adhesion of FHA-positive bacteria. Biochemical assays showed that this putative protein exhibited a dose-dependent binding affinity to heparan sulfate, indicating its adhesin activity. Moreover, ST-4526 showed increased antibiotic-resistance (nalidixic acid and fluoroquinolones) and a reduced ability for DNA uptake. Taken together, our data suggested that these combined features contributed to the clonal thriving of ST-4526 in Japan.

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<![CDATA[Chicken Immune Response after In Ovo Immunization with Chimeric TLR5 Activating Flagellin of Campylobacter jejuni]]> https://www.researchpad.co/article/5989dae1ab0ee8fa60bbbd40

Campylobacter jejuni is the main cause of bacterial food-borne diseases in developed countries. Chickens are the most important source of human infection. Vaccination of poultry is an attractive strategy to reduce the number of C. jejuni in the intestinal tract of chickens. We investigated the immunogenicity and protective efficacy of a recombinant C. jejuni flagellin-based subunit vaccine with intrinsic adjuvant activity. Toll-like receptor activation assays demonstrated the purity and TLR5 stimulating (adjuvant) activity of the vaccine. The antigen (20–40 μg) was administered in ovo to 18 day-old chicken embryos. Serum samples and intestinal content were assessed for antigen-specific systemic and mucosal humoral immune responses. In ovo vaccination resulted in the successful generation of IgY and IgM serum antibodies against the flagellin-based subunit vaccine as determined by ELISA and Western blotting. Vaccination did not induce significant amounts of flagellin-specific secretory IgA in the chicken intestine. Challenge of chickens with C. jejuni yielded similar intestinal colonization levels for vaccinated and control animals. Our results indicate that in ovo delivery of recombinant C. jejuni flagellin subunit vaccine is a feasible approach to yield a systemic humoral immune response in chickens but that a mucosal immune response may be needed to reduce C. jejuni colonization.

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<![CDATA[Production of Organic Acids by Probiotic Lactobacilli Can Be Used to Reduce Pathogen Load in Poultry]]> https://www.researchpad.co/article/5989d9e0ab0ee8fa60b6969d

Probiotic Lactobacillus can be used to reduce the colonization of pathogenic bacteria in food animals, and therefore reduce the risk of foodborne illness to consumers. As a model system, we examined the mechanism of protection conferred by Lactobacillus species to inhibit C. jejuni growth in vitro and reduce colonization in broiler chickens. Possible mechanisms for the reduction of pathogens by lactobacilli include: 1) stimulation of adaptive immunity; 2) alteration of the cecal microbiome; and, 3) production of inhibitory metabolites, such as organic acids. The Lactobacillus species produced lactic acid at concentrations sufficient to kill C. jejuni in vitro. We determined that lactic acid produced by Lactobacillus disrupted the membrane of C. jejuni, as judged by biophotonics. The spectral features obtained using Fourier-transform infrared (FT-IR) and Raman spectroscopy techniques were used to accurately predict bacterial viability and differentiate C. jejuni samples according to lactic acid treatment. FT-IR spectral features of C. jejuni and Lactobacillus grown in co-culture revealed that the metabolism was dominated by Lactobacillus prior to the killing of C. jejuni. Based on our results, the development of future competitive exclusion strategies should include the evaluation of organic acid production.

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<![CDATA[Estimating the prevalence of food risk increasing behaviours in UK kitchens]]> https://www.researchpad.co/article/5989db5fab0ee8fa60be1280

Foodborne disease poses a serious threat to public health. In the UK, half a million cases are linked to known pathogens and more than half of all outbreaks are associated with catering establishments. The UK Food Standards Agency (FSA) has initiated the UK Food Hygiene Rating Scheme in which commercial food establishments are inspected and scored with the results made public. In this study we investigate the prevalence of food risk increasing behaviours among chefs, catering students and the public. Given the incentive for respondents to misreport when asked about illegal or illicit behaviours we employed a Randomised Response Technique designed to elicit more accurate prevalence rates of such behaviours. We found 14% of the public not always hand-washing immediately after handling raw meat, poultry or fish; 32% of chefs and catering students had worked within 48 hours of suffering from diarrhoea or vomiting. 22% of the public admitted having served meat “on the turn” and 33% of chefs and catering students admitted working in kitchens where such meat was served; 12% of the public and 16% of chefs and catering students admitted having served chicken at a barbeque when not totally sure it was fully cooked. Chefs in fine-dining establishment were less likely to wash their hands after handling meat and fish and those who worked in award winning restaurants were more likely to have returned to work within 48 hours of suffering from diarrhoea and vomiting. We found no correlation between the price of a meal in an establishment, nor its Food Hygiene Rating Score, and the likelihood of any of the food malpractices occurring.

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<![CDATA[Campylobacter jejuni CsrA Regulates Metabolic and Virulence Associated Proteins and Is Necessary for Mouse Colonization]]> https://www.researchpad.co/article/5989dac6ab0ee8fa60bb2649

Campylobacter jejuni infection is a leading bacterial cause of gastroenteritis and a common antecedent leading to Gullian-Barré syndrome. Our previous data suggested that the RNA-binding protein CsrA plays an important role in regulating several important phenotypes including motility, biofilm formation, and oxidative stress resistance. In this study, we compared the proteomes of wild type, csrA mutant, and complemented csrA mutant C. jejuni strains in an effort to elucidate the mechanisms by which CsrA affects virulence phenotypes. The putative CsrA regulon was more pronounced at stationary phase (111 regulated proteins) than at mid-log phase (25 regulated proteins). Proteins displaying altered expression in the csrA mutant included diverse metabolic functions, with roles in amino acid metabolism, TCA cycle, acetate metabolism, and various other cell processes, as well as pathogenesis-associated characteristics such as motility, chemotaxis, oxidative stress resistance, and fibronectin binding. The csrA mutant strain also showed altered autoagglutination kinetics when compared to the wild type. CsrA specifically bound the 5’ end of flaA mRNA, and we demonstrated that CsrA is a growth-phase dependent repressor of FlaA expression. Finally, the csrA mutant exhibited reduced ability to colonize in a mouse model when in competition with the wild type, further underscoring the role of CsrA in C. jejuni colonization and pathogenesis.

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<![CDATA[Contribution of Amino Acid Catabolism to the Tissue Specific Persistence of Campylobacter jejuni in a Murine Colonization Model]]> https://www.researchpad.co/article/5989daddab0ee8fa60bbaa8b

Campylobacter jejuni is a major cause of food-borne disease in industrialized countries. Carbohydrate utilization by C. jejuni is severely restricted, and knowledge about which substrates fuel C. jejuni infection and growth is limited. Some amino acids have been shown to serve as carbon sources both in vitro and in vivo. In the present study we investigated the contribution of serine and proline catabolism to the in vitro and in vivo growth of C. jejuni 81-176. We confirmed that the serine transporter SdaC and the serine ammonia-lyase SdaA are required for serine utilization, and demonstrated that a predicted proline permease PutP and a bifunctional proline/delta-1-pyrroline-5-carboxylate dehydrogenase PutA are required for proline utilization by C. jejuni 81-176. C. jejuni 81-176 mutants unable to utilize serine were shown to be severely defective for colonization of the intestine and systemic tissues in a mouse model of infection. In contrast, C. jejuni 81-176 mutants unable to utilize proline were only defective for intestinal colonization. These results further emphasize the importance of amino acid utilization in C. jejuni colonization of various tissues.

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<![CDATA[The Prevalence of Campylobacter amongst a Free-Range Broiler Breeder Flock Was Primarily Affected by Flock Age]]> https://www.researchpad.co/article/5989daa6ab0ee8fa60ba7c62

Campylobacter successfully colonizes broiler chickens, but little is known about the longer term natural history of colonization, since most flocks are slaughtered at an immature age. In this study, the prevalence and genetic diversity of Campylobacter colonizing a single free-range broiler breeder flock was investigated over the course of a year. The age of the flock was the most important factor in determining both the prevalence and diversity of Campylobacter over time. There was no correlation with season, temperature, the amount of rain and sunshine, or the dynamics of colonization amongst geographically and temporally matched broiler flocks. The higher prevalence rates coincided with the age at which broiler chickens are typically slaughtered, but then in the absence of bio-security or other intervention methods, and despite changes in flock management, the prevalence fell to significantly lower levels for the remainder of the study. The genetic diversity of Campylobacter increased as the flock aged, implying that genotypes were accumulated within the flock and may persist for a long time. A better understanding of the ecology of Campylobacter within commercial chicken flocks will allow the design of more effective farm-based interventions.

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<![CDATA[Finnish Campylobacter jejuni Strains of Multilocus Sequence Type ST-22 Complex Have Two Lineages with Different Characteristics]]> https://www.researchpad.co/article/5989db33ab0ee8fa60bd249e

Background

Campylobacter jejuni is the major cause of human bacterial gastroenteritis worldwide, and in a minority of cases, post-infectious complications may occur. ST-22 complex (usually Penner serotype 19) strains have been overrepresented among patients with postinfectious complications of campylobacteriosis. We here present a characterization of a collection of 27 Finnish C. jejuni strains of ST-22 complex, from humans (22 strains) and animal sources (five strains), with the aim of contributing to our knowledge of the pathogenesis of C. jejuni infections.

Methodology/Principal Findings

All strains were analyzed by pulsed-field gel electrophoresis (PFGE) genotyping, lipo-oligosaccharide (LOS) locus class, Y-glutamyl transpeptidase (GGT) activity, in vitro biofilm formation ability, invasion and adhesion in HeLa cells and induction of IL-8 production. ST-22 complex contained five STs (ST-22; ST-1947; ST-1966; ST-3892; ST-3996) which were homogeneous in having sialylated LOS class A1 but on the other hand were distinguished into two major lineages according to the major STs (ST-22 and ST-1947) by different PFGE genotypes and certain other characteristics. All ST-22 strains had similar SmaI PFGE profiles, were GGT positive, and formed biofilms, except one strain, while ST-1947 strains were all GGT negative, did not form biofilm, had significantly higher motility than ST-22 (p<0.05) and had their SmaI PFGE profile. Invasion and adhesion as well as induction of IL-8 production on HeLa cells were strain-dependent characteristics.

Conclusions/Significance

ST-22 complex strains, reveal potential for molecular mimicry in host interactions upon infection as they all express sialylated LOS class A1. The two major STs, ST-22 and ST-1947 formed two homogeneous lineages, which differed from each other both phenotypically and genetically, suggesting that the strains may have evolved separately, perhaps by interacting with different spectra of hosts. Further studies are needed in order to understand if these two lineages are associated with different disease outcomes.

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<![CDATA[Genomic Characterization of the Guillain-Barre Syndrome-Associated Campylobacter jejuni ICDCCJ07001 Isolate]]> https://www.researchpad.co/article/5989db1bab0ee8fa60bce295

Campylobacter jejuni ICDCCJ07001 (HS:41, ST2993) was isolated from a Guillain-Barré syndrome (GBS) patient during a 36-case GBS outbreak triggered by C. jejuni infections in north China in 2007. Sequence analysis revealed that the ICDCCJ07001 genome consisted of 1,664,840 base pairs (bp) and one tetracycline resistance plasmid of 44,084 bp. The GC content was 59.29% and 1,579 and 37 CDSs were identified on the chromosome and plasmid, respectively. The ICDCCJ07001 genome was compared to C. jejuni subsp. jejuni strains 81-176, 81116, NCTC11168, RM1221 and C. jejuni subsp. doylei 269.97. The length and organization of ICDCCJ07001 was similar to that of NCTC11168, 81-176 and 81-116 except that CMLP1 had a reverse orientation in strain ICDCCJ07001. Comparative genomic analyses were also carried out between GBS-associated C. jejuni strains. Thirteen common genes were present in four GBS-associated strains and 9 genes mapped to the LOS cluster and the ICDCCJ07001_pTet (44 kb) plasmid was mosaic in structure. Thirty-seven predicted CDS in ICDCCJ07001_pTet were homologous to genes present in three virulence-associated plasmids in Campylobacter: 81-176_pTet, pCC31 and 81-176_pVir. Comparative analysis of virulence loci and virulence-associated genes indicated that the LOS biosynthesis loci of ICDCCJ07001 belonged to type A, previously reported to be associated with cases of GBS. The polysaccharide capsular biosynthesis (CPS) loci and the flagella modification (FM) loci of ICDCCJ07001 were similar to corresponding sequences of strain 260.94 of similar serotype as strain ICDCCJ07001. Other virulence-associated genes including cadF, peb1, jlpA, cdt and ciaB were conserved between the C. jejuni strains examined.

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<![CDATA[Risk Factors for Campylobacteriosis of Chicken, Ruminant, and Environmental Origin: A Combined Case-Control and Source Attribution Analysis]]> https://www.researchpad.co/article/5989daf4ab0ee8fa60bc267d

Background

Campylobacteriosis contributes strongly to the disease burden of food-borne pathogens. Case-control studies are limited in attributing human infections to the different reservoirs because they can only trace back to the points of exposure, which may not point to the original reservoirs because of cross-contamination. Human Campylobacter infections can be attributed to specific reservoirs by estimating the extent of subtype sharing between strains from humans and reservoirs using multilocus sequence typing (MLST).

Methodology/Principal Findings

We investigated risk factors for human campylobacteriosis caused by Campylobacter strains attributed to different reservoirs. Sequence types (STs) were determined for 696 C. jejuni and 41 C. coli strains from endemic human cases included in a case-control study. The asymmetric island model, a population genetics approach for modeling Campylobacter evolution and transmission, attributed these cases to four putative animal reservoirs (chicken, cattle, sheep, pig) and to the environment (water, sand, wild birds) considered as a proxy for other unidentified reservoirs. Most cases were attributed to chicken (66%) and cattle (21%), identified as the main reservoirs in The Netherlands. Consuming chicken was a risk factor for campylobacteriosis caused by chicken-associated STs, whereas consuming beef and pork were protective. Risk factors for campylobacteriosis caused by ruminant-associated STs were contact with animals, barbecuing in non-urban areas, consumption of tripe, and never/seldom chicken consumption. Consuming game and swimming in a domestic swimming pool during springtime were risk factors for campylobacteriosis caused by environment-associated STs. Infections with chicken- and ruminant-associated STs were only partially explained by food-borne transmission; direct contact and environmental pathways were also important.

Conclusion/Significance

This is the first case-control study in which risk factors for campylobacteriosis are investigated in relation to the attributed reservoirs based on MLST profiles. Combining epidemiological and source attribution data improved campylobacteriosis risk factor identification and characterization, generated hypotheses, and showed that genotype-based source attribution is epidemiologically sensible.

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<![CDATA[The Pathogenic Potential of Campylobacter concisus Strains Associated with Chronic Intestinal Diseases]]> https://www.researchpad.co/article/5989dae1ab0ee8fa60bbbde4

Campylobacter concisus has garnered increasing attention due to its association with intestinal disease, thus, the pathogenic potential of strains isolated from different intestinal diseases was investigated. A method to isolate C. concisus was developed and the ability of eight strains from chronic and acute intestinal diseases to adhere to and invade intestinal epithelial cells was determined. Features associated with bacterial invasion were investigated using comparative genomic analyses and the effect of C. concisus on host protein expression was examined using proteomics. Our isolation method from intestinal biopsies resulted in the isolation of three C. concisus strains from children with Crohn's disease or chronic gastroenteritis. Four C. concisus strains from patients with chronic intestinal diseases can attach to and invade host cells using mechanisms such as chemoattraction to mucin, aggregation, flagellum-mediated attachment, “membrane ruffling”, cell penetration and damage. C. concisus strains isolated from patients with chronic intestinal diseases have significantly higher invasive potential than those from acute intestinal diseases. Investigation of the cause of this increased pathogenic potential revealed a plasmid to be responsible. 78 and 47 proteins were upregulated and downregulated in cells infected with C. concisus, respectively. Functional analysis of these proteins showed that C. concisus infection regulated processes related to interleukin-12 production, proteasome activation and NF-κB activation. Infection with all eight C. concisus strains resulted in host cells producing high levels of interleukin-12, however, only strains capable of invading host cells resulted in interferon-γ production as confirmed by ELISA. These findings considerably support the emergence of C. concisus as an intestinal pathogen, but more significantly, provide novel insights into the host immune response and an explanation for the heterogeneity observed in the outcome of C. concisus infection. Moreover, response to infection with invasive strains has substantial similarities to that observed in the inflamed mucosa of Crohn's disease patients.

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<![CDATA[Campylobacter fetus Subspecies Contain Conserved Type IV Secretion Systems on Multiple Genomic Islands and Plasmids]]> https://www.researchpad.co/article/5989daf8ab0ee8fa60bc39f0

The features contributing to differences in pathogenicity of the Campylobacter fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode a type IV secretion system (T4SS) and fic domain (filamentation induced by cyclic AMP) proteins, which may disrupt host cell processes. In the genomes of 27 C. fetus strains, three phylogenetically-different T4SS-encoding regions (T4SSs) were identified: one was located in both the chromosome and in extra-chromosomal plasmids; one was located exclusively in the chromosome; and one exclusively in extra-chromosomal plasmids. We observed that C. fetus strains can contain multiple T4SSs and that homologous T4SSs can be present both in chromosomal genomic islands (GI) and on plasmids in the C. fetus strains. The GIs of the chromosomally located T4SS differed mainly by the presence of fic genes, insertion sequence elements and phage-related or hypothetical proteins. Comparative analysis showed that T4SS sequences, inserted in the same locations, were conserved in the studied C. fetus genomes. Using phylogenetic analysis of the T4SSs, it was shown that C. fetus may have acquired the T4SS regions from other Campylobacter species by horizontal gene transfer. The identified T4SSs and fic genes were found in Cff and Cfv strains, although the presence of T4SSs and fic genes were significantly associated with Cfv strains. The T4SSs and fic genes could not be associated with S-layer serotypes or geographical origin of the strains.

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<![CDATA[Genomic and phenotypic characteristics of Swedish C. jejuni water isolates]]> https://www.researchpad.co/article/5ab4d559463d7e0969868610

Campylobacter jejuni is the most common cause of bacterial gastroenteritis. Major reservoirs are warm-blooded animals, poultry in particular, but Campylobacter can also be transmitted via water. In this paper, we have taken a closer look at the biology and potential virulence of C. jejuni water isolates. Seven C. jejuni isolates from incoming surface water at water plants in Sweden were characterized with whole genome sequencing and phenotypical testing. Multi locus sequence typing analysis revealed that these isolates belonged to groups known to include both common (ST48CC) and uncommon (ST1275CC, ST683, ST793 and ST8853) human pathogens. Further genomic characterization revealed that these isolates had potential for arsenic resistance (due to presence of arsB gene in all isolates), an anaerobic dimethyl sulfoxide oxidoreductase (in three isolates) and lacked the MarR-type transcriptional regulator gene rrpB (in all but one isolate) earlier shown to be involved in better survival under oxidative and aerobic stress. As putative virulence factors were concerned, there were differences between the water isolates in the presence of genes coding for cytolethal distending toxin (cdtABC), Type VI secretion system and sialylated LOS, as well as in biofilm formation. However, all isolates were motile and could adhere to and invade the human HT-29 colon cancer cell line in vitro and induce IL-8 secretion suggesting potential to infect humans. This is, to the best of our knowledge, the first study where C. jejuni water isolates have been characterized using whole genome sequencing and phenotypical assays. We found differences and shared traits among the isolates but also potential to infect humans.

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<![CDATA[Influencing Factors and Applicability of the Viability EMA-qPCR for a Detection and Quantification of Campylobacter Cells from Water Samples]]> https://www.researchpad.co/article/5989daaaab0ee8fa60ba9190

In recent years, increasing numbers of human campylobacteriosis cases caused by contaminated water have been reported. As the culture-based detection of Campylobacter is time consuming and can yield false-negative results, the suitability of a quantitative real-time PCR method in combination with an ethidium monoazide pretreatment of samples (EMA-qPCR) for the rapid, quantitative detection of viable Campylobacter cells from water samples was investigated. EMA-qPCR has been shown to be a promising rapid method for the detection of viable Campylobacter spp. from food samples. Application of membrane filtration and centrifugation, two methods frequently used for the isolation of bacteria from water, revealed a mean loss of up to 1.08 log10 cells/ml from spiked samples. Both methods used alone lead to a loss of dead bacteria and accumulation of viable bacteria in the sample as shown by fluorescence microscopy. After filtration of samples, no significant differences could be detected in subsequent qPCR experiments with and without EMA pretreatment compared to culture-based enumeration. High correlations (R2 = 0.942 without EMA, R2 = 0.893 with EMA) were obtained. After centrifugation of samples, qPCR results overestimated Campylobacter counts, whereas results from both EMA-qPCR and the reference method were comparable. As up to 81.59% of nonviable cells were detected in pond water, EMA-qPCR failed to detect correct quantities of viable cells. However, analyses of spiked tap water samples revealed a high correlation (R2 = 0.863) between results from EMA-qPCR and the reference method. After membrane filtration, EMA-qPCR was successfully applied to Campylobacter field isolates, and results indicated an advantage over qPCR by analysing defined mixtures of viable and nonviable cells. In conclusion, EMA-qPCR is a suitable method to detect viable Campylobacter from water samples, but the isolation technique and the type/quality of the water sample impact the results.

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<![CDATA[The Natural Antimicrobial Carvacrol Inhibits Campylobacter jejuni Motility and Infection of Epithelial Cells]]> https://www.researchpad.co/article/5989d9f5ab0ee8fa60b7005c

Background

Natural compounds with anti-microbial properties are attractive reagents to reduce the use of conventional antibiotics. Carvacrol, the main constituent of oregano oil, inhibits the growth of a variety of bacterial foodborne pathogens. As concentrations of carvacrol may vary in vivo or when used in animal feed, we here investigated the effect of subinhibitory concentrations of the compound on major virulence traits of the principal bacterial foodborne pathogen Campylobacter jejuni.

Methods/Principal Findings

Motility assays revealed that subinhibitory concentrations of carvacrol inhibited the motility of C. jejuni without affecting bacterial growth. Immunoblotting and electron microscopy showed that carvacrol-treated C. jejuni still expressed flagella. The loss of motility was not caused by reduced intracellular ATP levels. In vitro infection assays demonstrated that subinhibitory concentrations of carvacrol also abolished C. jejuni invasion of human epithelial cells. Bacterial uptake of invasive Escherichia coli was not blocked by carvacrol. Exposure of C. jejuni to carvacrol prior to infection also inhibited cellular infection, indicating that the inhibition of invasion was likely caused by an effect on the bacteria rather than inhibition of epithelial cell function.

Conclusions/Significance

Bacterial motility and invasion of eukaryotic cells are considered key steps in C. jejuni infection. Our results indicate that subinhibitory concentrations of carvacrol effectively block these virulence traits by interfering with flagella function without disturbing intracellular ATP levels. These results broaden the spectrum of anti-microbial activity of carvacrol and support the potential of the compound for use in novel infection prevention strategies.

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